Purification Of Antitumor Protein From Trachyrhamphus Serratus And Investigation Of It's In Vitro Avtivity

The abounding resources of marine organisms provide more and newer compounds for finding antitumor medicine with high efficiency and low toxicity. The purification process, in vitro activity and antitumor mechanism of the antitumor protein from a marine animal, Trachyrhamphus serratus were investigated in this thesis.On the base of former studies, the crude protein was extracted from Trachyrhamphus serratus by using 40% ethanol extraction, macroporous absorption resin isolation, and ammonium sulfate precipitation. In this thesis, an antitumor active protein, named D2-1, was then purified with the methods of DEAE-Sepharose Fast Flow column chromatogramphy, Sephacryl-S100 chromatography. The purified protein D2-1 has main bands with 60 kDa and 35 kDa in relative molecular mass estimated by SDS—PAGE and needs further purification. Furthermore, the in vitro activity of D2-1 was investigated. The protein could inhibit the growth of tumor cell line A549, CCRF-CEM, LOVO, and L1210 in vitro with IC₅₀ of 0.30±0.04, 0.38±0.06, 1.15±0.18 and 1.63±0.14 mg/ml respectively. In comparison with IC₅₀ of Methotraexate (MTX) and Mitomycin (MMC) alone or combined with D2-1, the 72-hour IC₅₀ value of MTX in A549 cells decreased from 0.27±0.04μg/ml to 0.13±0.027μg/ml, and which of MMC, decreased from 0.09±0.02μg/ml to 0.016±0.004μg/ml (P>0.05) respectively. The 72-hour IC₅₀ values of MTX and MMC in CCRF-CEM cells decreased from 0.048±0.03μg/ml, 0.03±0.01μg/ml to 0.043±0.021μg/ml, 0.023±0.016μg/ml respectively. These results suggested that D2-1 have synergistic effects on inhibition of proliferation in A549 with MMC. Moreover, the study of antitumor mechamism showed that the protein purified in this thesis might induce the apoptosis of A549 in vitro...